Computational protocol: RNF8 and SCML2 cooperate to regulate ubiquitination and H3K27 acetylation for escape gene activation on the sex chromosomes

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Protocol publication

[…] Cells were suspended in chilled 1x PBS. One-eleventh volume of crosslinking solution (50 mM HEPES-NaOH pH 7.9, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 8.8% formaldehyde) was added to the cell suspension and incubated on ice for 8 min. One-twentieth volume of 2 M glycine was added to the cell suspension and incubated at room temperature for 5 min to stop the reaction. Cells were washed twice with PBS, frozen at -80°C, and lysed at 4°C for 10 min each in ChIP lysis buffer 1 (50 mM HEPES pH 7.9, 140 mM NaCl, 10% glycerol, 0.5% IGEPAL-630, 0.25% Triton X-100). After centrifugation at 2,000xg for 10 min at 4°C, pellets were resuspended with ChIP lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated at 4°C for 10 min. After centrifugation at 2,000xg for 10 min at 4°C, pellets were washed with TE containing 0.1% SDS and protease inhibitors (Sigma; 11836145001), and resuspended with the same buffer. Chromatin was sheared to approximately 200–500 bp by sonication using a Covaris sonicator at 10% duty cycle, 105 pulse intensity, 200 burst for 2 min. Sheared chromatin was cleared by centrifugation at 20,000xg for 20 min, followed by pre-incubation with Dynabeads Protein G. Chromatin immunoprecipitation was carried out on an SX-8X IP-STAR compact automated system (Diagenode). Briefly, Dynabeads Protein G were pre-incubated with 0.1% BSA for 2 h. Then, the cleared chromatin was incubated with beads conjugated to antibodies against H3K27ac (Active Motif; 39133) at 4°C for 8 h, washed sequentially with wash buffer 1 (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% NaDOC, and 1% Triton X-100), wash buffer 2 (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM EDTA, 0.1% SDS, 0.1% NaDOC, and 1% Triton X-100), wash buffer 3 (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NaDOC, and 0.5% NP-40), wash buffer 4 (10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.2% Triton X-100), and wash buffer 5 (10 mM Tris-HCl). DNA libraries were prepared through the ChIPmentation method []. Briefly, beads were resuspended in 30 μl of the tagmentation reaction buffer (10 mM Tris-HCl pH 8.0 and 5 mM MgCl2) containing 1 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) and incubated at 37°C for 10 min in a thermal cycler. The beads were washed twice with 150 μl cold wash buffer 1, incubated with elution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 250 mM NaCl, 0.3% SDS, 0.1 μg/μl Proteinase K) at 42°C for 30 min, and then incubated at 65°C for another 5 h to reverse cross-linking. DNA was purified with the MinElute Reaction Cleanup Kit (Qiagen) and amplified with NEBNext High-Fidelity 2x PCR Master Mix (NEB). Amplified DNA was purified by Agencourt AMPure XP (Beckman Coulter). Afterward, DNA fragments in the 250- to 500-bp size range were prepared by agarose gel size selection. DNA libraries were adjusted to 5 nM in 10 mM Tris-HCl pH 8.0 and sequenced with an Illumina HiSeq 2500.Data analysis was performed in the Wardrobe Experiment Management System (https://code.google.com/p/genome-tools/ []). For the analysis of H3K4me2, we used our publish data []. Briefly, reads were aligned to the mouse genome (mm10) with Bowtie (version 1.0.0 []) and displayed on a local mirror of UCSC genome browser as coverage. Islands of H3K27ac- and H3K4me2-enrichment were identified using MACS2 (version 2.0.10.20130712 []). MAnorm, software designed for the quantitative comparison of ChIP-seq datasets [], was applied to compare the enrichment profile of H3K27ac or H3K4me2 between pachytene spermatocytes and round spermatids. [...] The RNA-seq results of Rnf8;Scml2-dKO cells (two-biological replicates) were analyzed with our previous data obtained from wild-type, Rnf8-KO, and Scml2-KO mice (GSE55060, GSE69946) [, ]. Data analysis for RNA-seq was performed in the Wardrobe Experiment Management System. []. FASTQ files from the Illumina pipeline were aligned via the Spliced Transcripts Alignment to a Reference (STAR) software (version STAR_2.4.2a) [] with the following parameters:--outFilterMultimapNmax 1--outFilterMismatchNmax 2 (to see the full manual, go to this STAR GitHub page: https://github.com/alexdobin/STAR/blob/master/doc/STARmanual.pdf?raw=true). RefSeq annotation from the UCSC genome browser (11/2012) [] for the mm10 genome was used. The--outFilterMultimapNmax parameter was used to allow unique alignment only, and the--outFilterMismatchNmax parameter was used to allow a maximum of only 2 errors. All reads from resulting bam files were split for related isoforms with respect to RefSeq annotation. Then, an expectation-maximization algorithm was used to estimate appropriate numbers of reads for each isoform []. To estimate differences between experiments, the DESeq2 package [] was used. […]

Pipeline specifications

Software tools STAR, DESeq2
Application RNA-seq analysis
Organisms Homo sapiens